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Food, drug, insect sting allergy, and anaphylaxis
Peanut allergy: Effect of environmental peanut exposurein children with filaggrin loss-of-function mutations
Helen A. Brough, MSc, FRCPCH,a Angela Simpson, MD, PhD,b Kerry Makinson, MSc,a Jenny Hankinson, PhD,b
Sara Brown, MD,d Abdel Douiri, PhD,e Danielle C. M. Belgrave, MSc,b,c Martin Penagos, MD, MSc,a
Alick C. Stephens, PhD,a W. H. Irwin McLean, PhD, DSc, FRSE, FMedSci,d Victor Turcanu, PhD,a Nicolaos Nicolaou, MD,
PhD,b Adnan Custovic, MD, PhD,b* and Gideon Lack, MD, FRCPCHa* London, Manchester, and Dundee, United Kingdom
Background: Filaggrin (FLG) loss-of-function mutationslead to an impaired skin barrier associated with peanutallergy. Household peanut consumption is associatedwith peanut allergy, and peanut allergen in householddust correlates with household peanut consumption.Objective: We sought to determine whether environmentalpeanut exposure increases the odds of peanut allergy andwhether FLG mutations modulate these odds.Methods: Exposure to peanut antigen industwithin the first year oflife was measured in a population-based birth cohort. Peanutsensitization and peanut allergy (defined by using oral foodchallengesorcomponent-resolveddiagnostics [CRD])were assessedat 8 and 11 years. Genotyping was performed for 6FLGmutations.Results: After adjustment for infantile atopic dermatitis andpreceding egg skin prick test (SPT) sensitization, we found astrong and significant interaction between natural log (ln [loge])peanut dust levels andFLGmutations on peanut sensitization andpeanut allergy. Among children with FLGmutations, for each lnunit increase in the house dust peanut protein level, there was amore than6-fold increased odds of peanut SPT sensitization,CRD
From athe Department of Pediatric Allergy, Division of Asthma, Allergy and Lung
Biology, King’s College London and Guy’s and St. Thomas’ NHS Foundation Trust,
London; bthe Centre for Respiratory Medicine and Allergy, Institute of Inflammation
and Repair, Manchester Academic Health Sciences Centre, University of Manchester
and University Hospital of SouthManchester NHS Foundation Trust, Manchester; cthe
Centre for Health Informatics, Institute of Population Health, University of Manches-
ter; dthe Centre for Dermatology and Genetic Medicine, College of Life Sciences and
College of Medicine, Dentistry and Nursing, University of Dundee; and ethe Depart-
ment of Public Health Science, School of Medicine, King’s College London.
*These authors contributed equally to the manuscript and are joint senior authors.
The research was funded by Action Medical Research (S/P/4529) and supported by the
National Institute for Health Research (NIHR) Clinical Research Facility at Guy’s&St
Thomas’ NHS Foundation Trust and the NIHR Biomedical Research Centre based at
Guy’s and St Thomas’ NHS Foundation Trust and Kings College London. The views
expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or
the Department of Health. The Manchester Asthma and Allergy Study is supported by
Medical Research Council grants G0601361 and MR/K002449/1, the JP Moulton
Charitable Foundation, North West Lung Centre Charity, and the National Institute
for Health Research Clinical Research Facility at the University Hospital of South
Manchester NHS Foundation Trust. The Centre for Dermatology and Genetic Medi-
cine, University of Dundee, is funded by a Wellcome Trust Strategic Award
(098439/Z/12/Z; to W.H.I.M.). S.B. holds a Wellcome Intermediate Clinical Fellow-
ship (086398/Z/08/Z).
Disclosure of potential conflict of interest: H. A. Brough has received research support
from the Department of Health through the National Institute for Health Research
(NIHR) comprehensive Biomedical Research Centre award to Guy’s & St. Thomas’
NHS Foundation Trust in partnership with King’s College London and King’s College
Hospital NHS Foundation Trust and Action Medical Research, UK. A. Simpson has
received research support from the Medical Research Council, JP Moulton Charitable
Foundation, and the National Institute of Health Research. K. Makinson has received
research support from the Department of Health through the National Institute of
Health Research comprehensive Biomedical Research Centre award to Guy’s & St.
sensitization, or both in children at ages 8 years, 11 years, or bothand a greater than 3-fold increased odds of peanut allergycompared with odds seen in children with wild-type FLG. Therewas no significant effect of exposure in children without FLGmutations. In children carrying an FLG mutation, the thresholdlevel for peanut SPT sensitization was 0.92 mg of peanut proteinper gram (95% CI, 0.70-1.22 mg/g), that for CRD sensitizationwas 1.03 mg/g (95% CI, 0.90-1.82 mg/g), and that for peanutallergy was 1.17 mg/g (95% CI, 0.01-163.83 mg/g).Conclusion: Early-life environmental peanut exposure isassociated with an increased risk of peanut sensitization andallergy in children who carry an FLGmutation. These datasupport the hypothesis that peanut allergy developsthrough transcutaneous sensitization in childrenwith an impairedskin barrier. (J Allergy Clin Immunol 2014;134:867-75.)
Key words: FLG loss-of-function mutations, filaggrin, skin barrier,peanut sensitization, peanut allergy, environmental peanut exposure,dust, threshold
Thomas’ NHS Foundation Trust in partnership with King’s College London and
King’s College Hospital NHS Foundation Trust and the Immune Tolerance Network,
National Institutes of Health. S. Brown has received research support from the
Wellcome Trust Intermediate Clinical Fellowship and has received payment for
lectures from the American Academy of Allergy, Asthma & Immunology. A. Douiri
has received research support from the National Institute of Health Research. A. C.
Stephens has received research support from the Department of Health through the
NIHR comprehensive Biomedical Research Centre award to Guy’s & St. Thomas’
NHS Foundation Trust in partnership with King’s College London and King’s College
Hospital NHS Foundation Trust. W. H. I. McLean has received research support from
the Wellcome Trust. A. Custovic has consultant arrangements with Circassia; has
received research support from the Medical Research Council and the Moulton Char-
itable Foundation; and has received payment for lectures from GlaxoSmithKline,
Thermo Fisher Scientific, Novartis, and ALK-Abell�o. G. Lack has received research
support from the Department of Health through the NIHR comprehensive Biomedical
Research Centre award to Guy’s & St. Thomas’ NHS Foundation Trust in partnership
with King’s College London and King’s College Hospital NHS Foundation Trust and
Action Medical Research, UK; is a board member of DBV Technologies; has consul-
tant arrangements with the Anaphylaxis Campaign and the National Peanut Board; has
received payment for lectures from Sodilac, Novartis, Nestle Nutrition, GlaxoSmith-
Kline, and the Serono Symposia International Foundation; and has stock/stock options
with DBV Technologies. The rest of the authors declare that they have no relevant
conflicts of interest.
Received for publicationApril 2, 2014; revised August 20, 2014; accepted for publication
August 20, 2014.
Corresponding author: Gideon Lack, MD, Children’s Allergy Unit, 2nd Floor, Stairwell
B, South Wing, Guy’s and St Thomas’ NHS Foundation Trust, Westminster Bridge
Road, London SE1 7EH, United Kingdom. E-mail: [email protected].
0091-6749/$36.00
� 2014 American Academy of Allergy, Asthma & Immunology
http://dx.doi.org/10.1016/j.jaci.2014.08.011
867
J ALLERGY CLIN IMMUNOL
OCTOBER 2014
868 BROUGH ET AL
Abbreviations used
AD: A
topic dermatitisCRD: C
omponent-resolved diagnosticsFLG: F
ilaggrinGEE: P
enalized generalized estimating equations methodologyISU: IS
AC standardized unitLLQ: L
ower limit of quantitationMAAS: M
anchester Asthma and Allergy StudyOFC: O
ral food challengeOR: O
dds ratiosIgE: A
llergen-specific IgESPT: S
kin prick testThere is a clear association between early-onset atopicdermatitis (AD) and food allergy.1,2 Children with AD have animpaired skin barrier, which might allow antigen to penetratethe skin and sensitize the subject.3,4 In children with a historyof AD, 90% of those who went on to have peanut allergy hadbeen exposed topically to creams containing Arachis species(peanut) oil in the first 6 months of life.2 In mice epicutaneousexposure to food allergens after skin stripping induces a potentallergic TH2-type response associated with high IL-4, IL-5, andallergen-specific IgE (sIgE) levels and systemic anaphylaxis afteroral challenge.5,6
Filaggrin is responsible for the strength and integrity of thestratum corneum7 and regulates the permeability of the skin towater and antigens.8 Loss-of-function mutations in the geneencoding filaggrin (FLG) are present in up to 50% of patientswith moderate-to-severe AD9,10 and have been shown to increasethe risk of inhalant allergic sensitization, allergic rhinitis,asthma,11,12 and peanut allergy.13 In the flaky tail mouse, whichhas a 1-bp deletion mutation (5303delA) within the murine flggene (analogous to common human FLG loss-of-functionmutations), topical allergen application leads to cellularinfiltration and allergen-specific antibody response, even withoutskin stripping.14 This suggests that filaggrin deficiency, even inthe absence of dermatitis, might be sufficient for transcutaneoussensitization.
High consumption of peanut by household members during thechild’s first year of life is associated with an increased risk ofpeanut allergy, possibly because of environmental peanutexposure in the child’s home15; however, in this studyquestionnaire-based assessment of household peanut consump-tion was not validated against an objective measure of peanut inthe environment and was potentially subject to retrospectivebias. We recently showed that peanut protein in household dustis positively correlated with household peanut consumption.16
In addition, we showed that peanut protein in dust activatesbasophils from children with peanut allergy in a dose-dependent manner and is thus biologically active.16
We hypothesized that peanut sensitization can occur throughpresentation of environmental peanut antigen through animpaired skin barrier to underlying antigen-presenting cells. Toaddress this hypothesis, we investigated whether early-lifeenvironmental peanut exposure measured directly by quantifyingpeanut antigen in household dust was a risk factor for thedevelopment of peanut allergy and whether this relationshipwas modified by FLG genotype. Specifically, we predicted that anincrease in the peanut protein concentration in household dust
during infancy would be associated with an increase in school-age peanut sensitization and allergy and that this effect wouldbe augmented in children with 1 or more FLG loss-of-functionmutations.
METHODS
Study populationThe Manchester Asthma and Allergy Study (MAAS) is an unselected birth
cohort described in detail elsewhere (registration: ICRCTN72673620).17 In
brief, 1184 subjects were recruited prenatally from 1995 to 1997 and followed
up at ages 1, 3, 5, 8, and 11 years. The study was approved by the local ethics
committee; parents provided written informed consent.
Data sourcesValidated questionnaires were interviewer administered to collect infor-
mation on parentally reported symptoms and physicians’ diagnoses. Parental
report of a history of AD during infancy was assessed by using a modified
International Study of Asthma and Allergies in Childhood questionnaire to
apply the UK Working Party’s diagnostic criteria for AD.18 Peanut sensitiza-
tion was assessed at ages 8 and 11 years by using skin prick tests (SPTs) to
whole peanut extract (Hollister-Stier, Spokane, Wash)19 and by measuring
sIgE to whole peanut extract and peanut components Ara h 1, 2, and 3 with
ImmunoCAP (age 8 years) or the ISAC Multiplex Immuno Solid-phase
Allergen Chip (age 11 years; Thermo Fisher Scientific, Uppsala, Sweden).20
Maternal peanut consumption during pregnancy and breast-feeding were
collected retrospectively (aged 8 years) in a subset of patients assessed for
peanut allergy by means of diagnostic oral food challenge (OFC).
Definition of outcomesPeanut SPT sensitization. Peanut SPT sensitization was defined
as a mean wheal diameter of 3 mm or greater than that elicited by the negative
control.
Peanut component-resolved diagnostics sensitiza-
tion. Peanut component-resolved diagnostics (CRD) sensitization was
defined as sIgE to the peanut components Ara h 1, 2, or 3 of 0.35 kUA/L or
(8 years) or 0.35 ISAC standardized units (ISU) or greater (11 years).20
Patients with Ara h 1, 2, or 3 levels of less than 0.35 kUA/L (8 years) or
0.35 ISU (11 years) were deemed non-CRD sensitized. If no CRD analysis
was available, then patients with peanut sIgE levels of less than 0.2 kUA/L
ImmunoCAP were considered not CRD sensitized.
Peanut allergy. All children with evidence of peanut sensitization atage 8 years (peanut SPT response >_3 mm or sIgE level >_0.2 kUA/L) were
offered an OFC to peanut to determine allergy versus tolerance.19 Open
OFCs were applied among children who had a history of tolerating peanut
on consumption; all other children underwent a double-blind, placebo-
controlled OFC.19 OFC results were considered positive after development
of 2 or more objective signs indicating an allergic reaction.19 Children with
a convincing history of an immediate hypersensitivity reaction on exposure
to peanut combined with a peanut sIgE level of 15 kUA/L or greater,21 an
SPT response of 8 mm or greater,22 or both (age 8 years) were considered to
have peanut allergy and did not undergo an OFC. Two children with a
convincing history of an immediate hypersensitivity reaction on exposure to
peanut and an SPT response of 3 mm or greater who refused consent for
OFCs were considered to have peanut allergy based on an Ara h 2 level of
0.35 ISU or greater19 at subsequent follow-up at age 11 years.
Quantitation of environmental peanut exposure in
household dustDust sampleswere collected predominantly at 36weeks’ gestation from the
lounge-sofa, as previously described.23 If no antenatal dust sample was
available from the lounge-sofa, then dust samples from 6 or 12 months were
analyzed for peanut protein (where available). Dust samples were extracted
TABLE I. Demographics and clinical characteristics of the included group (n 5 623) versus the excluded group (n 5 561) and
whole group (n 5 1184)
Included group*
(n 5 623)
Excluded groupy(n 5 561)
Whole group
(n 5 1184)
P value,
included
(n = 623)
vs excluded
(n = 561)Total no. No. (%) Total no. No. (%) Total no. No. (%)
Peanut SPT sensitization at 8 y 559 30 (5.4) 360 18 (5.0) 920 48 (5.2) .69
Peanut SPT sensitization at 11 y 450 19 (4.2) 256 13 (5.1) 706 32 (4.5) .41
Peanut SPT sensitization at age 8 and/or 11 y§ 434 35 (8.1) 237 24 (10.1) 710 59 (8.3) .15
Peanut CRD sensitization at age 8 y 371 13 (3.5) 211 7 (3.3) 584 20 (3.4) .84
Peanut CRD sensitization at age 11 y 297 12 (4.0) 154 8 (5.2) 451 20 (4.4) .37
Peanut CRD sensitization at age 8 and/or 11 y§ 241 19 (7.9) 116 9 (7.8) 357 28 (7.8) .94
Peanut allergy at age 8 and/or 11 y 577 20 (3.5) 382 10 (2.6) 959 30 (3.1) .19
History of AD during infancy 614 207 (33.7) 477 190 (39.8) 1091 397 (36.4) <.01
No AD on clinical assessment at age 1 y 338 272 (80.5) 173 142 (82.1) 511 414 (81.0) .46
Mild AD on assessment at age 1 y 338 46 (13.6) 173 25 (14.5) 511 71 (13.9) .66
Moderate/severe AD at age 1 y 338 20 (5.9) 173 6 (3.5) 511 26 (5.1) .01
Combined FLG loss-of-function mutation 623 57 (9.1) 234 29 (12.4) 857 86 (10.0) .02
Parental report of ‘‘hay fever ever’’ in the child 569 135 (23.7) 400 105 (26.3) 969 240 (24.8) .18
Egg SPT sensitization at age 3 y 545 21 (3.9) 398 15 (3.8) 943 36 (3.8) .92
Male sex 623 311 (49.9) 561 331 (59.0) 1184 642 (54.2) <.001
Full older siblings (same mother and father) 623 316 (50.7) 532 297 (55.8) 1155 614 (53.2) .02
Parental atopy (low vs medium/high risk) 621 501 (80.7) 514 443 (86.2) 1135 944 (83.2) .001
Breast-feeding (yes vs no) 618 443 (71.7) 497 337 (67.8) 1115 780 (70.0) .03
Peanut consumption during pregnancy (yes vs no) 70 56 (80.0) 41 35 (85.4) 111 91 (82.0) .28
Peanut consumption during breast-feeding (yes vs no) 59 45 (76.3) 29 24 (82.8) 88 69 (78.4) .26
House dust mite reduction measures� 160 88 (55.0) 93 45 (48.4) 253 133 (52.6) 1.00
Maternal age at baseline (y), mean (SD) 615 30.67 (4.74) 499 30.02 (4.81) 1114 30.38 (4.78) .51
Peanut protein in dust (mg/g) using values below
LLQ, median (IQR)
623 0.73 (0.40-1.33) 128 0.78 (0.36-1.40) 751 0.73 (0.38-1.35) .96
Peanut protein in dust (mg/g) using LLQ/2,
median (IQR)
623 0.73 (0.25-1.33) 128 0.78 (0.25-1.40) 751 0.73 (0.25-1.35) .90
IQR, Interquartile range.
*Included group comprised of white children enrolled in MAAS with available sofa dust within the first year of life and successful FLG genotyping.
�Children were excluded for the following reasons: (1) nonwhite ethnicity, (2) lack of available blood sample for FLG genotyping or failed genotyping, or (3) no dust extract
available for the assessment of environmental peanut allergen exposure.
�‘‘High-risk’’ infants (both parents with positive SPT responses) with no pets in the home in MAAS were randomized to house dust mite reduction measures versus control
subjects.
§Children who were not peanut sensitized at age 8 or 11 years and missing data at the other time point were classed as having missing sensitization data.
J ALLERGY CLIN IMMUNOL
VOLUME 134, NUMBER 4
BROUGH ET AL 869
in borate-buffered saline (0.1% Tween 20, pH 8.0) and stored at 2208C until
analysis. Peanut protein in dust extracts was determined by using the Veratox
polyclonal ELISA against whole peanut protein (Neogen, Lansing, Mich),
which has been validated for sensitivity, specificity, and reliability in
measuring peanut protein contamination of food,24,25 dust, and wipe
samples.26 The Veratox ELISA lower limit of quantitation (LLQ) for peanut
protein in dust was 100 ng/mL (0.5 mg/g based on a dust sample weighing
between 50-100 mg); this variable was analyzed by using a fixed calculation
for values of less than this level (LLQ/2; results are shown in Table E1 in
this article’s Online Repository at www.jacionline.org)27 and by using all
data of less than this value (results in the main body of the article) because
the variable with LLQ/2 created 230 (37%) censored data points.28 Analyses
for both forms of the peanut dust variable were compared to determine
whether the 2 different ways of dealing with data of less than the LLQ
made a material difference to the results obtained. Participant information
was blinded from the researcher performing the ELISA-based dust analyses.
GenotypingFLG genotyping was performed with probes and primers, as previously
described.9 Genotyping for R501X, S3247X, and R2447X loss-of-function
mutations was performed with a TaqMan-based allelic discrimination assay
(Applied Biosystems, Cheshire, United Kingdom). Mutation 2282del4 was
genotyped by sizing of a fluorescently labeled PCR fragment on a 3100 or
3730 DNA sequencer. FLGmutations 3673delC and 3702delG were assessed
by means of GeneScan analysis of fluorescently labeled PCR products. These
6 FLG mutations have been consistently associated with AD in white
populations10; however, because some of these FLG mutations are not found
in nonwhite subjects,29 all nonwhite participants were excluded from analyses
that included FLG genotype. Data were analyzed as combined carriage of an
FLG null allele; that is, if a child carried 1 or more of the 6 genetic variations,
he or she was considered an FLG null allele carrier. Complete FLG genotype
results (ie, results for all 6 FLG loss-of-function mutations screened) were
available for 805 (76.0%) of 1059 white participants, 117 samples failed
genotype analysis for 1 or more mutations, and no sample was available in
137 participants. In cases with incomplete FLG data, the presence of 1 FLG
mutation defined that case as a carrier; participants with incomplete
genotyping data in whom all successfully tested alleles were wild-type alleles
were excluded from further analysis because their FLG genotype status
remained ambiguous.
Statistical analysisData were analyzed with STATA 12.1 software (StataCorp, College
Station, Tex). Demographics and clinical characteristics were compared
between participants and nonparticipants. Count data were compared by using
the Pearson x2 test. Continuous data were compared with the Student t test for
normally distributed data and the Mann-Whitney U test for nonnormally
distributed data. All variables except maternal age and peanut protein in
dust were compared by using the Pearson x2 test. Maternal age was normally
FIG 1. CONSORT diagram outlining participant flow. Peanut allergy outcomes are highlighted in boxesoutlined in boldface. DBPCFC, Double-blind, placebo-controlled food challenge.
J ALLERGY CLIN IMMUNOL
OCTOBER 2014
870 BROUGH ET AL
distributed and thus was compared with the Student t test. Peanut protein in
dust (without natural log [ln] transformation) was not normally distributed
and thus was compared with the Mann-Whitney U test. Peanut protein in
dust (inmicrograms per gram) underwent ln transformation for subsequent an-
alyses. Factors associated with peanut allergy at the ages of 8 years, 11 years,
or both were assessed by using a penalized logistic regression methodology to
account for unbalanced data (20/577 had peanut allergy).30 Factors associated
with peanut sensitization (SPT and CRD results) were assessed by using
penalized generalized estimating equations methodology (GEE) through a
quasi–least squares approach, with an exchangeable working correlation ma-
trix to account for repeated measures within subjects at 8 and 11 years.31
Goodness of fit of the GEE statistical model was assessed by using the
quasilikelihood under independence model criterion. The goodness of fit of
the penalized logistic regression methodology statistical model was assessed
by using the Akaike information criterion. We tested whether the effect of
environmental peanut exposure on peanut sensitization and allergy was
modified by FLG genotype by including an interaction term.
The additive effect of FLG loss-of-function mutation was calculated by
using the exponential of the coefficient (b) of the interaction (FLG genotype
by peanut dust exposure) minus the baseline coefficient (b) of peanut dust
exposure. The predictive probability of peanut sensitization and allergy was
calculated from the multivariate regression model. Threshold levels of peanut
protein in dust for peanut sensitization and allergy were calculated by
using the intersection between wild-type FLG versus FLG mutation in the
multivariate regression model.30,32 To evaluate the reliability of the thresholds
obtained and the uncertainty around them, we conducted bootstrap cross-
validation with 1000 replications.
RESULTS
Participants and descriptive dataDetails of the participant flow are presented in Fig 1. From
1184 participants, we analyzed data from 623 white childrenwith available FLG genotyping and early-life environmental pea-nut exposure. Of these children, at age 8 years, 32 had no peanutSPT or peanut sIgE data, 70 were peanut sensitized (of these, 3children were sensitized at age 5 years and had no peanut SPTor sIgE data at age 8 years), 1 was not peanut sensitized butreported a reaction on peanut exposure, and 520 were not peanutsensitized and reported no reactions to peanut (of these, 1 was
subsequently peanut sensitized at age 11 years and thus impos-sible to classify). Seven children with a convincing history ofan allergic reaction on peanut exposure and a peanut sIgE levelof 15 kUA/L or greater, an SPT response of 8 mm or greater, orboth were classified as having peanut allergy; the remaining 64sensitized children were invited for an OFC (29 double-blind,placebo-controlled food challenges and 35 open challenges).We were unable to contact 1 subject, and 14 refused consent (ofthese, 2 were classified as having peanut allergy at age 11 yearson the basis of a convincing history of an immediate hypersensi-tivity reaction on exposure to peanut and an Ara h 2 level >_0.35ISU). Thus 20 children were defined as having peanut allergy,557 were defined as nonallergic, and 46 could not be classified(because of missing SPT and sIgE data or because they declinedconsent for an OFC).
The demographics of the whole group, both included andexcluded children, are shown in Table I. Comparison of theincluded and excluded groups revealed no differences in peanutsensitization or allergy; we observed small (but statistically sig-nificant) differences in parental atopy, FLG status, history andseverity of AD, sex, breast-feeding, and sibship position. FLGloss-of-function mutations were carried by 57 (9.1%) of 623 chil-dren (all children; Table I) and 4 (20%) of 20 children with peanutallergy (Table II). A history of infantile AD was present in 207(33.7%) of 614 (all children) children and 16 (80%) of 20 childrenwith peanut allergy. Of the 16 children with peanut allergy withwild-type FLG, 13 (81%) had a history of infantile AD. The me-dian peanut protein concentration in dust was 0.73 mg/g (inter-quartile range, 0.40-1.33 mg/g); the peanut allergen level wasless than the LLQ in 230 (36.9%) of 623 homes.
FLG genotype modifies the effect of early-life
environmental peanut on the risk of peanut
sensitization and allergyFactors associated with both peanut sensitization and
peanut allergy were history and severity of infantile AD, FLG
TABLE II. FLG genotype frequencies in 20 children with peanut allergy and 577 children without peanut allergy at ages 8 years, 11
years, or both
R501X 2282del4 S3247X R2447X 3673delC 3702delG
Combined FLG
loss-of-function genotype
No. (%) of peanut allergic children with FLG genotype (n 5 20)
Wild-type FLG 18 (90.0) 17 (85.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 16 (80.0)
FLG loss-of-function mutation 2 (10.0) 3 (15.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 4 (20.0)*�Failure of analysis� 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)
No. (%) of children without peanut allergy with FLG genotype (n 5 557)
Wild-type FLG 533 (95.7) 535 (96.05) 552 (99.1) 551 (98.9) 603 (100.0) 603 (100.0) 507 (91.0)
FLG loss-of-function mutation 23 (4.1) 20 (3.6) 4 (0.7) 5 (0.9) 0 (0.0) 0 (0.0) 50 (9.0)*§
Failure of analysis� 1 (0.2) 2 (0.35) 1 (0.2) 1 (0.2) 0 (0.0) 0 (0.0)
*There were no mutant allele homozygotes for any allele tested.
�This includes 1 compound heterozygote (R501X/2282del4).
�Although individual FLG genotypes failed, if a child had incomplete data but had a mutant FLG allele, they were included as a case in the combined loss-of-function genotype. If
they had incomplete data but all alleles successfully tested were wild-type alleles, they were excluded because this could indicate a false-negative result.
§This includes 2 compound heterozygotes (R501X/2282del4).
TABLE III. Clinical and demographic factors associated with peanut SPT and CRD sensitization and peanut allergy on univariate
GEE and penalized logistic regression methodology analysis
Peanut SPT sensitization
adjusted for age at
assessment (8 1 11 y;
GEE; n 5 584)
Peanut CRD sensitization
adjusted for age at
assessment (8 1 11 y;
GEE; n 5 437)
Peanut allergy at age 8 y, 11
y,
or both (LR; n 5 577)
OR 95% CI P value OR 95% CI P value OR 95% CI P value
History of AD during infancy 10.5 4.2-26.1 <.001 11.9 3.3-43.1 <.001 8.9 2.9-26.9 <.001
AD severity, no AD at 1 y Reference category Reference category Reference category
Mild AD on assessment at 1 y 2.2 0.6-8.4 .25 3.4 0.7-16.5 .13 5.0 1.1-23.2 .04
Moderate-to-severe AD at 1 y 20.8 4.1-62.4 <.001 16.6 3.2-86.6 .001 28.0 6.6-118.8 <.001
Combined FLG loss-of-function mutations 3.5 1.5-8.3 <.01 4.0 1.4-11.4 <.01 2.5 0.8-7.9 .11
Parental report of ‘‘hay fever ever’’ in the child 3.4 1.6-7.3 .001 3.4 1.3-9.2 .02 4.2 1.6-11.1 <.01
Egg SPT sensitization at age 3 y 12.3 4.5-33.6 <.001 16.4 4.8-56.0 <.001 25.5 8.4-77.0 <.001
Male sex 2.2 1.0-4.6 .04 1.8 0.7-4.8 .22 1.6 0.6-3.9 .33
Full older siblings (same mother and father) 0.9 0.4-1.8 .72 0.5 0.2-1.4 .19 0.7 0.3-1.8 .46
Parental atopy, low vs medium/high risk 6.9 0.9-51.4 .06 1.9 0.4-8.3 .42 4.7 0.6-35.5 .13
Breast-feeding (yes vs no) 1.0 0.5-2.2 .99 2.7 0.6-11.9 .19 1.6 0.5-4.8 .43
Peanut consumption during pregnancy (yes vs no) 1.0 0.3-2.8 .93 0.8 0.2-2.8 .72 0.5 0.2-1.9 .32
Peanut consumption during breast-feeding (yes vs no) 0.8 0.3-2.3 .65 0.8 0.2-2.7 .70 0.6 0.2-2.0 .38
House dust mite reduction measures 1.0 0.3-3.2 .95 0.8 0.2-4.4 .81 0.7 0.1-2.6 .57
Maternal age at baseline (y) 1.0 1.0-1.1 .31 1.1 1.0-1.1 .06 1.0 0.9-1.1 .79
Peanut protein in dust (ln transformed mg/g)* 1.3 0.9-1.7 .16 1.2 0.8-1.8 .33 1.2 0.8-1.8 .47
Age at assessment (8 or 11 y) 0.8 0.5-1.1 .10 1.0 0.7-1.5 1.00 NA NA NA
Values in boldface are significant.
LR, Penalized logistic regression methodology; NA, not applicable.
*Peanut protein in dust: values less than the LLQ were used in this analysis.
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loss-of-function mutation (trend for allergy), egg SPT sensitiza-tion at age 3 years, and parental report of ‘‘hay fever ever’’ inthe child on univariate analysis (Table III). Peanut protein levelsin dust were not associated with peanut sensitization or allergyoverall; however, there was a strong and significant interactionon univariate analysis between FLG genotype and early-lifeenvironmental peanut exposure on peanut SPT sensitization(odds ratio [OR], 5.3; 95% CI, 1.8-15.3; P < .01) and peanutCRD sensitization (OR, 4.5; 95% CI, 1.5-13.5; P < .01)and a trend toward peanut allergy (OR, 2.7; 95% CI, 0.9-8.0;P5 .07) (Table IV). Given the low number of children with pea-nut allergy outcomes, wewere conservative in the selection of co-variates in the multivariate model and used 2 covariates (egg SPTsensitization at age 3 years and a history of infantile AD) that wereboth highly associated with peanut SPT/CRD sensitization and
allergy. In the multivariate analysis, with the inclusion of an inter-action variable for FLG genotype*(ln peanut exposure), we founda strong and significant change in FLG genotype divergence withearly-life environmental peanut exposure on both peanut sensiti-zation and allergy (Table IV). These interactions were consistentfor peanut SPT sensitization (OR, 5.2; 95%CI, 2.1-13.1;P <.001;Fig 2, A), peanut CRD sensitization (OR, 5.3; 95% CI, 1.9-14.8;P5 .001; Fig 2, B), and clinically confirmed peanut allergy (OR,3.2; 95% CI, 1.1-9.8; P5 .04; Fig 3). Analysis of the peanut dustvariable with LLQ/2 did not show a material difference in results(see Table E1). The additive effect of each ln unit increase inhouse dust peanut in children with 1 or more FLG loss-of-function mutations was 6.1-fold for peanut SPT sensitization,6.5-fold for peanut CRD sensitization, and 3.3-fold for peanut al-lergy in the multivariate model. In children with a wild-type FLG
TABLE IV. GEE for peanut sensitization using quasilikelihood under independent model criterion goodness-of-fit analyses
GEE peanut SPT sensitization
adjusted for clustering at
age 8 1 11 y (n 5 584)
GEE peanut CRD sensitization
adjusted for clustering at
age 8 1 11 y (n 5 437)
LR for peanut allergy at
age 8 y, 11 y, or both (n 5 577)
No.* OR 95% CI P value QIC§ No.y OR 95% CI P value QIC§ No.z OR 95% CI P value AIC§
Combined FLG loss-of-function
mutation
584 3.5 1.5-8.3 <.01 386.6 437 4.0 1.4-11.4 <.01 215.3 577 2.54 0.82-7.88 .11 175.6
Age at assessment (8 or 11 y) 0.8 0.5-1.1 .10 0.9 0.6-1.4 .69 NA
Combined FLG loss-of-function
mutation
584 3.6 1.5-8.2 <.01 386.7 437 4.0 1.4-11.0 <.01 216.5 577 2.5 0.8-7.9 .11 177.1
Peanut protein in dust
(ln transformed mg/g)k1.3 0.9-1.7 .15 1.2 0.8-1.8 .27 1.2 0.8-1.8 .46
Age at assessment (8 or 11 y) 0.7 0.5-1.1 .10 0.9 0.6-1.4 .66 NA
Combined FLG loss-of-function
mutation
584 2.4 0.7-8.6 .17 370.7 437 2.6 0.6-11.0 .20 207.9 577 2.2 0.6-8.2 .23 175.6
Peanut protein in dust
(ln transformed mg/g)k0.9 0.6-1.3 .52 0.8 0.5-1.4 .38 0.9 0.6-1.6 .82
Interaction FLG*peanut in dust 5.3 1.8-15.3 <.01 4.5 1.5-13.5 <.01 2.70 0.9-8.0 .07
Age at assessment (8 or 11 y) 0.7 0.5-1.1 .10 0.9 0.6-1.4 .66 NA
Combined FLG loss-of-function
mutation
516 1.8 0.4-7.5 .41 303.9 396 1.3 0.2-7.6 .78 176.7 511 1.1 0.3-5.2 .87 132.5
Peanut protein in dust
(ln transformed mg/g)k0.9 0.6-1.3 .50 0.8 0.5-1.5 .53 0.98 0.5-1.9 .98
Interaction FLG*peanut in dust 6.8 2.6-17.5 <.001 6.6 2.3-18.9 <.001 3.9 1.3-11.8 .02
Egg SPT sensitization at age 3 y 16.2 4.5-59.0 <.001 25.1 5.2-122.1 <.001 34.84 9.9-122.4 <.001
Age at assessment (8 or 11 y) 0.7 0.5-1.1 .14 0.9 0.6-1.6 .82 NA
Combined FLG loss-of-function
mutation
516 1.1 0.3-5.2 .87 279.4 396 1.0 0.2-5.5 .95 167.6 511 0.8 0.2-3.9 .83 129.3
Peanut protein in dust
(ln transformed mg/g)k0.9 0.6-1.3 .45 0.8 0.5-1.4 .46 1.0 0.5-1.8 .95
Interaction FLG*peanut in dust 5.2 2.1-13.1 <.001 5.3 1.9-14.8 .001 3.2 1.1-9.8 .04
Egg SPT sensitization at age 3 y 8.8 2.2-34.5 <.01 13.0 2.3-75.3 <.01 19.95 5.4-74.0 <.001
History of AD during infancy 7.5 2.4-23.2 <.001 5.4 1.2-24.2 .03 4.04 1.2-14.1 .03
Age at assessment (8 or 11 y) 0.7 0.4-1.1 .12 1.0 0.6-1.7 .90 NA
Values in boldface are significant.
AIC, Akaike information criterion; LR, penalized logistic regression methodology; NA, not applicable; QIC, quasilikelihood under independent model criterion.
*��White children enrolled in MAAS with available sofa dust within the first year of life, successful FLG genotyping, and peanut SPT* or CRD� sensitization or peanut allergy�assessment.
§Reductions in quasilikelihood under independent model criterion (GEE) and Akaike information criterion (LR) values denote improved goodness of fit of the statistical model.
kPeanut protein in dust: values less than the LLQ were used in this analysis.
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872 BROUGH ET AL
genotype, there was no association between early-life environ-mental peanut exposure and subsequent peanut sensitization orallergy.
Threshold environmental peanut levels in dust for
peanut sensitization and allergyIn children carrying 1 or more FLG loss-of-function mutations,
the threshold environmental peanut allergen level for peanut SPTsensitization was20.079 ln transformed units (0.92 mg of peanutprotein/gram of dust; 95% CI, 0.70-1.22 mg/g), that for CRDsensitization was 0.032 ln transformed units (1.03 mg/g; 95%CI, 0.90-1.82 mg/g), and that for peanut allergy was 0.156 lntransformed units (1.17 mg/g; 95% CI, 0.01-163.83 mg/g).
DISCUSSIONThis study demonstrates a gene-environment interaction on the
development of peanut sensitization and clinically proven peanutallergy. In children carrying 1 or more FLG loss-of-function
mutations, there was a dose-response relationship betweenearly-life environmental exposure to peanut protein in householddust and subsequent peanut sensitization and allergy; each ln unit(2.7-fold) increase in house dust peanut exposure during infancywas associated with a more than 6-fold increase in the odds ofschool-age peanut sensitization and a 3.3-fold increase in theodds of school-age peanut allergy. Therefore we demonstrated aconsistent interaction between FLG genotype and peanut dustexposure for peanut SPT sensitization, major allergen sensitiza-tion, and clinically proven peanut allergy. Previous studies havealso shown a stronger effect of FLG loss-of-function mutationson peanut sensitization than peanut allergy.33 The interactionbetween FLG genotype and environmental peanut exposure wassignificant after adjusting for infantile AD and preceding eggsensitization; thus the modifying effect of FLG genotype wasindependent of AD or other atopy markers.
Among FLG mutation carriers, peanut protein levels in dustreached a maximum of 14.78 mg/g; thus an increase in peanutdust exposure from the LLQ (0.5 mg/g) to 14.78 mg/g equated
FIG 2. Mean predictive probability of peanut sensitization over 8 and 11
years on GEE analysis with increasing environmental peanut exposure
(defined by ln transformed peanut protein in micrograms per gram of dust)
for children with 1 or more FLG loss-of-function mutations versus those
with wild-type FLG. The model was adjusted for a history of infantile AD
and egg SPT sensitization at age 3 years. Interaction ORs and 95% CIs dis-
played between peanut protein in dust and FLG loss-of-function mutations
on peanut sensitization are shown. Predictive probability is only shown
within the observable environmental peanut exposure data obtained.
A, Peanut SPT sensitization. B, Peanut CRD sensitization.
FIG 3. Mean adjusted predictive probability of peanut allergy at 8 years, 11
years, or both on multivariate penalized logistic regression analysis with
increasing environmental peanut exposure (defined by ln transformed
peanut protein in micrograms per gram of dust) in children with 1 or
more FLG loss-of-function mutations versus those with wild-type FLG.
Interaction ORs and 95% CIs are displayed between peanut protein in
dust and FLG loss-of-function mutations on peanut allergy. Predictive
probability is only shown within the observable environmental peanut
exposure data obtained.
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to an almost 30-fold increase (3.4 ln scales), which is equivalent toa 58-fold (3.33.4) increase in the odds of peanut allergy. These re-sults suggest that the level of early-life environmental peanutexposure in children who carry FLG loss-of-function variantsmight critically influence the development of peanut sensitizationand, importantly, clinical peanut allergy; however, future work isrequired to ensure the linearity of peanut protein data over theentire range of peanut protein in dust. In contrast, no associationwas seen between environmental peanut exposure and peanutsensitization or allergy in children without FLG mutations. Inchildren carrying an FLG mutant allele, the mean thresholdpeanut protein level in dust for peanut sensitization and allergywas around twice the LLQ of the ELISA (0.50 mg/g). Thus onthe basis of our findings in this white United Kingdom population,minimal quantities of peanut protein in the environment couldlead to peanut sensitization and allergy in children who carryFLG loss-of-function mutations, but the risk markedly increaseswith increasing exposure.
Previous studies have shown gene-environment interactionsbetween FLG loss-of-function mutations and other atopic dis-eases.34 Among children carrying an FLGmutation, those whosefamilies owned a cat had an approximately 4-fold odds of havingAD compared with those whose families did not own a cat; therewas no effect of cat ownership among children without FLGmutations.34 Contact allergy to nickel is twice as common inadults with the FLG frameshift mutation 2282del4,35 and inmurine models flg loss-of-function mutations lead to increasedbidirectional paracellular penetration of water-soluble tracersand reduced inflammatory threshold to allergens.36 There is asignificant association between FLG mutations and developmentof asthma and allergic sensitization but only in children withpreceding AD.37 This has been used as an argument for the roleof FLG loss-of-function mutations as a predisposing factor forallergic sensitization after epicutaneous exposure to allergens.Peanut protein in environmental dust and surfaces could penetratedisrupted skin because of impaired filaggrin production and couldbe taken up by Langerhans cells, leading to a TH2 response andIgE production by B cells.38,39 Studies are investigating the roleof thymic stromal lymphopoietin produced by keratinocytes inresponse to environmental antigens in patients with AD.40
Thymic stromal lymphopoietin in combination with enhancedallergen penetration through a damaged epidermis could lead toa TH2-type milieu; it would be interesting to review this in thecontext of filaggrin-deficient children with high levels of environ-mental peanut exposure.
There are certain limitations to this study. We were unable toinclude all MAAS participants because of the availability ofearly-life dust samples and FLG genotyping. Because the 6 FLGloss-of-function mutations assessed have been associated withAD in white populations,12 we excluded all nonwhite partici-pants. Given that 95% of MAAS participants were white, this isunlikely to lead to bias. On comparing the groups of includedversus excluded children, there were some small differences intheir demographic characteristics, but importantly, there were
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874 BROUGH ET AL
no significant differences in peanut sensitization or allergy rates;therefore these are unlikely to have influenced the results. Peanutallergen levels in lounge-sofa dust might not be the best index ofinfant exposure; however, we have shown previously that there ishigh within-home correlation of peanut protein levels in dust,particularly between an infant’s bed and play area.26 In ourpreviously published work the infant play area was usually inthe lounge, which was also the location of the sofa in theMAAS study. There were no available data on the amount of pea-nut the infant was consuming; however, given that the majority ofdust collected was antenatal, these peanut dust levels would nothave been due to the infant consuming peanut.
We acknowledge that there are small numbers of subjects withconfirmed peanut allergy in whom FLG genotype and early-lifepeanut exposure data are available. This reflects the complexitiesof measuring all necessary predictors over the life course inchildren with robustly ascertained clinical outcomes that arethemselves relatively uncommon (FLG loss-of-function muta-tions and clinical peanut allergy). We emphasize that the findingsof an interaction between FLG loss-of-function genotype andenvironmental peanut exposure for sensitization (howevermeasured) and peanut allergy are consistent, in keeping with pre-vious gene-environment interactions for FLG, and biologicallyplausible.
It is important to consider how peanut allergen in dust mightlead to sensitization to assess the clinical applicability of ourfindings; although this might lead to epicutaneous sensitizationthrough direct skin contact, we cannot exclude the possibility ofinhalation of dust particles containing peanut allergen. Althoughfilaggrin is not expressed in the lung41 or inferior nasal turbi-nates,42 it is expressed in the cornified epithelium in the vestibularnasal lining.11 However, several studies suggest that peanut ispoorly aerosolizeable26,43 and report that allergic symptoms afterinhalation of peanut have not been replicated on blinded chal-lenges.44 It is also important to determine how peanut proteingets into household dust. Peanut protein is present on hand wipesand in saliva up to 3 hours after peanut consumption and thusmight be amenable to transfer through this route.26 Fox et al15
found that household consumption of peanut butter was morehighly associated with peanut allergy in infants than householdconsumption of covered forms of peanut-containing foods.They hypothesized that peanut butter was more likely to lead tosensitization through hand-to-hand contact because it is stickyand thus more likely to be transferred onto surfaces (and dust)or people. Peanut protein persists on table surfaces and sofa-pillow dust, despite usual cleaning measures,26 and thus mightbe an important source of exposure.
Although our study focused on peanut sensitization and allergy,FLG loss-of-function mutations might confer susceptibility toenvironmental exposure to other food allergens in dust, such asfish, egg, and cow’s milk.45 The dual-allergen-exposure hypothe-sis postulates that food allergy develops through transcutaneousexposure to allergen through a disrupted skin barrier, whereasoral exposure leads to tolerance induction.38 Our findings of adose-response effect for peanut allergen in dust on the develop-ment of peanut allergy in children genetically predisposed to askin barrier defect support this hypothesis. Furthermore, ourstudy raises the intriguing possibility of identifying a group ofchildren with FLG loss-of-function mutations and targetingthem in interventional studies through early environmentalmodification.
We thankMrs L. Campbell, Molecular Medicine, University of Dundee, for
developing the TaqMan assay conditions and Professor A. Grieve, PhD, Aptiv
Solutions, for his statistical help. We also thank the children and their parents
in MAAS for their continued support and enthusiasm. We greatly appreciate
the commitment they have given to the project. Finally, we acknowledge the
hard work and dedication of the MAAS study team (research fellows, nurses,
physiologists, technicians, and clerical staff).
Clinical implications: Children with FLG loss-of-functionmutations are at an increased risk of peanut sensitization andallergy if they are exposed to peanut antigen in householddust in early life. Interventional studies to assess a causalrelationship are required.
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TABLE E1. GEE for peanut sensitization using the quasilikelihood under independent model criterion goodness-of-fit analyses
GEE for peanut SPT sensitization
adjusted for clustering at
age 8 and 11 y (n 5 584)
GEE for peanut CRD sensitization
adjusted for clustering at
age 8 and 11 y (n 5 437)
LR for peanut allergy at
age 8 y, 11 y, or both (n 5 577)
No.* OR 95% CI P value QIC§ No.y OR 95% CI P value QIC§ No.z OR 95% CI P value AIC§
Combined FLG loss-of-function
mutation
584 3.5 1.5-8.3 <.01 386.6 437 4.0 1.4-11.4 <.01 215.3 577 2.5 0.8-7.9 .11 175.6
Age at assessment (8 or 11 y) 0.8 0.5-1.1 .10 0.9 0.6-1.4 .69 NA
Combined FLG loss-of-function
mutation
584 3.6 1.5-8.2 <.01 386.5 437 3.9 1.4-10.9 <.01 215.9 577 2.6 0.8-7.9 .11 176.8
Peanut protein in dust
(ln transformed mg/g)k1.2 0.9-1.7 .15 1.3 0.90-1.8 .19 1.2 0.8-1.8 .35
Age at assessment (8 or 11 y) 0.7 0.5-1.1 .10 0.9 0.6-1.4 .65 NA
Combined FLG loss-of-function
mutation
584 2.8 0.8-9.8 .17 373.2 437 2.6 0.6-11.1 .20 208.2 577 2.3 0.6-8.4 .21 175.5
Peanut protein in dust
(ln transformed mg/g)k0.9 0.6-1.3 .58 0.9 0.5-1.4 .51 1.0 0.6-1.6 .97
Interaction FLG*peanut in dust 4.3 1.4-12.8 .01 4.0 1.4-11.4 .01 2.5 0.9-7.1 .08
Age at assessment (8 or 11 y) 0.7 0.5-1.1 .10 0.9 0.6-1.4 .66 NA
Combined FLG loss-of-function
mutation
516 2.1 0.5-8.4 .30 307.1 396 1.2 0.2-7.2 .78 177.0 511 1.1 0.2-5.1 .91 132.4
Peanut protein in dust
(ln transformed mg/g)k0.9 0.6-1.3 .59 0.9 0.6-1.5 .75 1.1 0.6-1.9 .83
Interaction FLG*peanut in dust 5.5 2.0-14.7 .001 6.0 2.2-16.2 <.001 3.7 1.3-10.7 .02
Egg SPT sensitization at age 3 y 15.9 4.4-57.8 <.001 26.3 5.3-130.2 <.001 35.9 10.1-127.7 <.001
Age at assessment (8 or 11 y) 0.7 0.5-1.1 .13 0.9 0.6-1.6 .81 NA
Combined FLG loss-of-function
mutation
516 1.3 0.3-5.8 .71 279.4 396 0.9 0.2-5.3 .93 167.9 511 0.8 0.2-3.9 .81 129.3
Peanut protein in dust
(ln transformed mg/g)k0.9 0.6-1.3 .52 0.9 0.6-1.4 .67 1.0 0.6-1.8 .89
Interaction FLG*peanut in dust 4.3 1.7-11.0 <.01 4.8 1.8-12.6 <.01 3.1 1.1-8.9 .04
Egg SPT sensitization at age 3 y 8.6 2.2-33.6 <.01 13.5 2.3-79.2 <.01 20.4 5.5-76.3 <.001
Atopic eczema during infancy 7.6 2.5-23.3 <.001 5.4 1.2-24.2 <.01 4.1 1.2-14.1 .03
Age at assessment (8 or 11 y) 0.7 0.4-1.1 .12 1.0 0.6-1.7 .91 NA
Values in boldface are significant.
AIC, Akaike information criterion; LR, penalized logistic regression methodology; NA, not applicable; QIC, quasilikelihood under independent model criterion.
*��White children enrolled in the MAAS with available sofa dust within the first year of life, successful FLG genotyping, and peanut SPT* or CRD� sensitization or peanut
allergy� assessment.
kPeanut protein levels in dust less than the LLQ were assigned an LLQ/2 calculation.
§Reductions in QIC (GEE) and AIC (LR) values denote improved goodness-of-fit of statistical model.
J ALLERGY CLIN IMMUNOL
OCTOBER 2014
875.e1 BROUGH ET AL